We have recently developed a microfluidic perfusion and imaging platform for large-scale detection of the response of dissociated mouse olfactory sensory neurons (OSNs) to various odorants; we refer to this platform as smell-on-a-chip platform. The overall goal of this project is to further develop the existing smell-on-a-chip platform to increase automation so as to improve data quality and increase overall experimental throughput. Our goal is to characterize the dynamics and the specificity of the responses of OSNs to a large variety of odorants, complex odors, and pheromones. Pheromones are volatile organic compounds that elicit or modulate innate behaviors such as mating, rearing of young, aggression and territory marking. In mammals, pheromones are primarily detected by the vomeronasal organ (VNO), but the olfactory epithelium (OE) also appears to play a role in pheromone detection because (in rodents) pheromone-associated behaviors and activation of the olfactory bulb (OB) are still present even if the VNO is removed. Although the VNO in humans has no detectable connection to the brain, the role of pheromone-sensitive OSNs in the mouse OE strongly suggests that pheromone signaling in humans occurs through the OE. OB activation is clearly a proof that there are olfactory sensory neurons (OSNs) in the OE that participate in pheromonal detection, yet the detection of these OSNs by traditional methods has proven elusive, likely because they exist in very low numbers. Hence, identification of these pheromone-specialist OSNs in the OE will require high-throughput detection methods. In this proposal we will utilize our smell-on-a-chip platform to detect the responses of dissociated mouse OSNs to known odorants, odors and pheromones. By imaging thousands of OSNs simultaneously we will be able to find rarely-occurring OSN responses. The detection and isolation of pheromone-specialist mouse OSNs would open the way for single-cell gene expression studies in mice towards a deeper, molecular-level understanding of pheromonal-induced behaviors in humans. The characterization of pheromonal responses at the cellular and molecular level is of paramount importance for understanding a large number of innate behaviors (such as sexual attraction, mother-child bonding, and menstrual cycle synchronization, to name only a few), and are of vital interest to the perfume and food industries. The successful completion of this project would also provide a platform of general applicability in a variety of fields for measuring the behavior of a large number of single cells, such as in toxicity studies, drug testing, and small-molecule screening (e.g. for cancer biomarkers), and in finding cells with rare, pathological behaviors from a large population of normally-behaving cells (e.g. the presence of tumorigenic cells in the bloodstream). Obtaining rich, single-cell statistics allows for discerning uniquely-responsive sub-populations of cells and for determining intrinsic cell behavior variability. In the proposed study, we will apply the smell-on-a-chip platform to screen OSNs, but the platform has broad applicability to any imaging-based, high-throughput screen of single dissociated cells in large numbers. Furthermore, rare cells (amongst an array of >28,000 microwells, only one cell per well) can be singled out and manually retrieved for further analysis (e.g. PCR amplification) after characterizing their response to known compounds.